Team:NCTU Formosa/project

Overview

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PBAN

Introduction

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Design of Red promoter

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Reference

Temperature-regulated system

Introduction

It is not a dream anymore that "making the impossible possible" with the increase of modern technology. It is also a serious problem that what human has brought to Earth along with our progressing civilization. Drastic environmental changes are inevitable due to the fact of massive developing industries. Temperature changing are one of the problems we need to confront.

Cells are constantly subjected to changing environmental conditions. Comparing with chemical substances, temperature is more accessible, convenient and lower costing. Moreover, temperature is also less burden to the organisms. Therefore, few organisms use temperature as a regulating factor. A mechanism found in different organisms that makes the cell respond to thermal changes, for example, is the RNA thermometer.

An RNA thermometer is a temperature-sensitive non-coding RNA molecule which regulates gene expression. The expression of heat-shock, cold-shock and some virulence genes are coordinated in response to temperature changes. There are several systems suggested in references that are based on RNA secondary structure. This structural transition can then expose or occlude important regions of RNA such as a ribosome binding site, which then affects the translation rate of a nearby protein-coding gene, so we can say that the RNA thermometers are just like riboswitches.

Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is also a widely used regulatory strategy.

Mechanism

RNA thermometers are identified in the 5' UTR of messenger RNA, upstream of a protein-coding gene.

Unlike normal RBS, the 37 °C RBS has a unique or rather stable hairpin structure which shaped like a lock. If the temperature drops below 37 °C, the base-pairing neucleotides on the RNA will form a stable secondary hairpin structure, occluding the Shine-Dalgarno sequence (SD sequence) thus disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded within. Since the structure is sustained by base pairing, heat can be employed to break the hydrogen bonds. So when it reaches 37 °C, the bonds would be broken and the hairpin would be unfolded, causing the SD sequence to be exposed and permitting the binding of the ribosome, which then starts the translation. This means that by raising temperature to a certain threshold, the 37 °C RBS can function as a normal RBS. In this way, gene expression can be regulated on the RNA level by temperature.

Design

The specific ribosome binding site (RBS) [http://parts.igem.org/Part:BBa_K115002 BBa_ K115002] is a great RNA thermometer. It has high translation activity at high temperature (> 37 °C) and low translation activity at room temperature. It was designed for the temperature-dependent genetic circuit in E. coli, with a green fluorescent protein (GFP) used as the reporter protein.

Originally, it is in the 5'-UTR of the Salmonella agsA gene, which codes for a small heat shock protein that can be used for temperature sensitive post-transcriptional regulation, initiating translation at 37 °C. Transcription of the agsA gene is controlled by the alternative sigma factor σ32. Additional translational control depends on a stretch of four uridines that pair with the SD sequence, so they are named "FourU". They repress translation of this protein by base-pairing with the Shine-Dalgardo sequence of the mRNA. This prevents ribosomes from binding the start codon.

In our project, we use the 37 °C RBS in both red light induced circuit and dark response circuit and it is related to the production of GFP and BFP. We use this 37 °C RBS to design our project because we want to use different conditions to produce different substances. Therefore, the characteristic and sensitivity of 37 °C RBS perfectly fits our purpose which we want to regulate our system through temperature.

Reference

Small RNA-regulated system

Introduction

Base pairing offers a powerful way for one RNA to control the activity of another. Both prokaryotes and eukaryotes have many cases which a single-stranded RNA base pairs with a complementary region of an mRNA. As a result, it prevents expression of the mRNA.

RNA interference (RNAi), also called post transcriptional gene silencing, is a process in which RNA molecules inhibit gene expression by destroying specific mRNA molecules. In 2006, Andrew Fire and Craig C.Mello shared the Nobel Prize in Physiology or Medicine because of their study on RNA interference¹¹. This powerful gene silencing tool in eukaryotes has been used in many research. As their counterparts in bacteria, small non-coding RNAs (sRNAs) are important regulatory roles.

Small RNAs (sRNAs) have become increasingly significant in playing the role of bacterial gene regulation. Most sRNAs interact with the targeted mRNAs by imperfect base pairing, reducing the translation efficiency and recruiting chaperones such as Hfq for translation termination. The sRNAs would bind to the target with its hairpin-like structure which wound by some of it's own sequences, and the chaperons would stuck between the hairpins in order to protect the sRNA-mRNA combination from degrading. In the end, the sRNAs regulate gene expression by forestalling translation. Since this regulated-system has already been employed in vivo, it is necessary to design an artificial sRNA that targets specifically to a desired genes, so that we can prevent the sRNA from affecting other undesired genes.

Unlike the regulated-system popular used in iGEM projects (e.g. P_tet & tetR, P_lux & LuxR, etc.), sRNA regulated-system seems more efficient. Because the inhibition works under RNA level, which means the energy wasted in E. coli is less than other system (producing proteins to regulate).

Figure 8 depicts how the small RNA chaperone, Hfq, associates with the small RNA and represses a target mRNA.¹²

Mechanism

How do small RNAs regulate gene expression? Figure 9 shows the mechanism of repressing one target mRNA. The small RNA has three stem-loop double stranded RNA structures, and the loop which is closest to the 3’ terminus is complementary to the sequence preceding the initiation codon of the target mRNA. Base pairing between small RNA and the target mRNA prevents the ribosome from binding to the initiation codon, so the translation would be repressed.^13,14

Design

The following is the process of our idea in designing the sRNA.

We have designed two artificial sRNAs, each modified from different paper. The sRNA-1 is based on afsRNA ARlacZ1, which is created by Hongmarn Park and his colleagues.¹⁵ SibC is a small RNA sequence which is already found in E.coli. Hongmarn Park and his colleagues changed few base pairs of SibC to make ARlacZ1, purposed to make the secondary structure more stable. After that, they tried to change 9 different locations of the target-recognition sequence to test the effect on gene silencing. Our design is to modify from the RNA sequence which has the best efficiency : ARlacZ7. In accordance with afsRNA ARlacZ7, we changed the target-recognition sequence to finally complete our sRNA-1.

The other sRNA sequence we produced is called sRNA-2. It is employed in this project selected from a library of artificial sRNA that was constructed by fusing a randomized antisense domain of Spot42, the scaffold that is known for recruiting the RNA chaperons. The sRNA we picked contains a consensus sequence, 5’-CCCUC-3’, which can base pair with the SD sequence due to complementary binding. This sRNA, as expected, effectively regulates gene expression by reducing translation efficiency and recruiting Hfq. In addition, this sRNA shows high specificity against its targeted gene, OmpF, as it doesn't hold significant activity against other genes from E. coli genome (Sharma and others, 2011).

The sRNA picked is competent, but we hoped that it can target any desired gene. Therefore, we designed a RBS by employing the sRNA targeting region from OmpF and making the AUG codon sufficiently apart from the SD sequence for ribosome binding. By adding this RBS to the upstream of any desired gene, the gene can be regulated by sRNA.

Reference

Cover image credit: DVQ